Given the risks associated with antimicrobial resistance and its link with antimicrobial use, available data on antimicrobial use in the Belgian pig, broiler and veal calf production were compared. Allowing for comparison of the data available from three peer-reviewed scientific articles, the unit of measurement for antimicrobial use was the Treatment Incidence (TI), defined as the number of animals per 1000 treated daily with one 'defined' (DDDA) or 'used daily dose animal' (UDDA). Moreover, extrapolation of farm-level data to national-level data was attempted according to the European Surveillance of Veterinary Antimicrobial Consumption (ESVAC) methodology, to estimate the amount of antimicrobials used in Belgium per species. Although, among the three species, the highest TI was observed in veal calves (TIDDDA=414, TIUDDA=379), based on the extrapolation, it was estimated that most antimicrobials were administered to pigs (159.4 tons). Thus, the most rapid decline in the total use could potentially be achieved by targeting the pig sector. During the process of data collection for comparison and calculation, it became obvious that there is a need for harmonized monitoring programs.

Het voorkomen van resistentie tegen antimicrobiële middelen houdt tal van risico’s in. Bovendien is er een link tussen het voorkomen van resistentie tegen antimicrobiële middelen en het gebruik van deze middelen. Het doel van deze studie is de beschikbare data over het gebruik van antimicrobiële middelen in de Belgische varkens-, vleeskuikens- en vleeskalversector te vergelijken. Om de beschikbare data van drie peer-reviewed, wetenschappelijke artikelen te kunnen vergelijken, werd het gebruik uitgedrukt in behandelingsincidenties (BI), gedefinieerd als het dagelijks aantal behandelde dieren per 1000 dieren met 1 ‘defined’ (DDDA) of ‘used daily dose animal’ (UDDA). Daarnaast werd getracht de data over het gebruik op bedrijfsniveau te extrapoleren naar het gebruik op nationaal niveau op basis van de methodologie van de European Surveillance of Veterinary Antimicrobial Consumption (ESVAC). Zo werd een schatting beoogd van de totale hoeveelheid antimicrobiële middelen die gebruikt worden in België per diersector. Voor de drie sectoren werd de hoogste behandelingsincidentie waargenomen in de vleeskalversector (TIDDDA=414, TIUDDA=379). Bij de schatting, gebaseerd op extrapolatie, werd echter verondersteld dat de grootste hoeveelheid antimicrobiële middelen toegediend werden aan varkens (159.4 ton). De snelste daling in de totale hoeveelheid antimicrobiële middelen kan dus wellicht bereikt worden door middel van acties in de varkenssector. Tijdens het collecteren van de data om berekeningen en vergelijkingen voor deze studie mogelijk te maken, werd het duidelijk dat er dringend nood is aan geharmoniseerde monitoringsprogramma’s.

It is believed that biosecurity influences production figures and health status of pig herds, nevertheless, few studies succeed in demonstrating and quantifying this relation. In the present study, 95 randomly selected Belgian closed or semi-closed pig herds were visited to quantify the biosecurity status of the herd by means of a biosecurity scoring system (Biocheck) with a range from 0 (= total absence of biosecurity) to 100 (= perfect biosecurity). At the same visit additional data concerning herd, farmer, production characteristics and the use of antimicrobials was collected. The Antimicrobial use was quantified by calculating treatment incidences (TI). The external biosecurity (preventing pathogens from entering the herd) ranged between 45 and 89/100 with an average of 65/100, whereas the internal biosecurity (reducing within herd spread of infection) score was on average 52/100 (min 18; max 87). The number of sows and fattening pigs on a herd ware positively associated with the external biosecurity score. This indicates that on larger herds, more attention is paid to biosecurity. A negative association was seen between the internal biosecurity score and the age of the buildings as well as with the years of experience of the farmer. This indicates that biosecurity is higher on the agenda with younger farmers and in more modern herds.

The use of antimicrobials in production animals has become a worldwide concern in the face of rising resistance levels in commensal, pathogenic and zoonotic bacteria. In the years 2007 and 2008 antimicrobial consumption records were collected during two non consecutive production cycles in 32 randomly selected Belgian broiler farms. Antimicrobials were used in 48 of the 64 monitored production cycles, 7 farms did not use any antimicrobials in both production cycles, 2 farms only administered antimicrobials in one of the two production cycles, the other 23 farms applied antimicrobial treatment in both production cycles. For the quantification of antimicrobial drug use, the treatment incidences (TI) based on the defined daily doses (the dose as it should be applied: DDD) and used daily doses (the actual dose applied: UDD) were calculated. A mean antimicrobial treatment incidence per 1000 animals of 131.8 (standard deviation 126.8) animals treated daily with one DDD and 121.4 (SD 106.7) animals treated daily with one UDD was found. The most frequently used compounds were amoxicillin, tylosin and trimethoprim-sulphonamide with a mean TIUDD of 37.9, 34.8, and 21.7, respectively. The ratio of the UDD/DDD gives an estimate on correctness of dosing. Tylosin was underdosed in most of the administrations whereas amoxicillin and trimethoprim-sulphonamide were slightly overdosed in the average flock.

The monitoring of antimicrobial use is an essential step to control the selection and spread of antimicrobial resistance. Between January and October 2010 data on prophylactic and metaphylactic antimicrobial use were collected retrospectively on 50 closed or semi-closed pig herds. Ninety-three percent of the group treatments were prophylactic whereas only 7% were methaphylactic. The most frequently used antimicrobials orally applied at group level were colistin (30.7%), amoxicillin (30.0%), trimethoprim-sulfonamides (13.1%), doxycycline (9.9%) and tylosin (8.1%). The most frequently applied injectable antimicrobials were tulathromycin (45.0%), long acting ceftiofur (40.1%) and long acting amoxicillin (8.4%). The treatment incidences (TI) based on the used daily dose pig (UDDpig or the actually administered dose per day per kg pig of a drug) for all oral and injectable antimicrobial drugs was on average 200.7 per 1000 pigs at risk per day (min = 0, max = 699.0), while the TI based on the animal daily dose pig (ADDpig or the national defined average maintenance dose per day per kg pig of a drug used for its main indication) was slightly higher (average = 235.8, min = 0, max = 1322.1). This indicates that in reality fewer pigs were treated with the same amount of antimicrobials than theoretically possible. Injectable products were generally overdosed (79.5%), whereas oral treatments were often underdosed (47.3%). In conclusion, this study shows that prophylactic group treatment was applied in 98% of the visited herds and often includes the use of critically important and broad-spectrum antimicrobials. In Belgium, the guidelines for prudent use of antimicrobials are not yet implemented.

Acquired resistance of Escherichia coli to 3rd generation cephalosporin antimicrobials is a relevant issue in intensive broiler farming. In Belgium, about 35% of the E. coli strains isolated from live broilers are resistant to 3rd generation cephalosporins while over 60% of the broilers are found to be carrier of these 3rd generation cephalosporin resistant E. coli (CREC) after selective isolation. A model aimed at estimating the exposure of the consumer to CREC by consumption of broiler meat was elaborated. This model consists of different modules that simulate the farm to fork chain starting from primary production, over slaughter, processing and distribution to storage, preparation and consumption of broiler meat. Input data were obtained from the Belgian Food Safety agencies' annual monitoring plan and results from dedicated research programs or surveys. The outcome of the model using the available baseline data estimates that the probability of exposure to 1000 colony forming units (cfu) of CREC or more during consumption of a meal containing chicken meat is ca. 1.5%, the majority of exposure being caused by cross contamination in the kitchen. The proportion of CREC (within the total number of E. coli) at primary production and the overall contamination of broiler carcasses or broiler parts with E. coli are dominant factors in the consumer exposure to CREC. The risk of this exposure for human health cannot be estimated at this stage given a lack of understanding of the factors influencing the transfer of cephalosporin antimicrobial resistance genes from these E. coli to the human intestinal bacteria and data on the further consequences of the presence of CREC on human health.

OBJECTIVES:To document and quantify drug use in white veal calves, an intensive livestock production system where multidrug resistance is abundantly present.METHODS:Drug consumption data were prospectively collected on 15 white veal production cohorts (n = 5853 calves) in Belgium (2007-09). Treatment incidences (TIs) based on animal defined daily dose (ADD), prescribed daily dose (PDD) and used daily dose (UDD) were calculated. Risk factors were identified by linear regression.RESULTS:The average TI(ADD) of antimicrobial treatments was 416.8 ADD per 1000 animals at risk. Predominantly, oral group antimicrobial treatments were used (95.8%). Of the oral group antimicrobial treatments, 12% and 88% were used for prophylactic or metaphylactic indications, respectively. The main indication for group and individual drug use was respiratory disease. The most frequently used antimicrobials (group treatments) were oxytetracycline (23.7%), amoxicillin (18.5%), tylosin (17.2%) and colistin (15.2%). Deviations from the leaflet dosage recommendations were frequently encountered, with 43.7% of the group treatments underdosed (often oxytetracycline and tylosin to treat dysbacteriosis). In 33.3% of the oral antimicrobial group treatments a combination of two antimicrobial preparations was used. Smaller integrations used more antimicrobials in group treatments than larger ones (P < 0.05); an integration is defined as a company that combines all steps of the production chain by having its own feed plant and slaughterhouse and by placing its calves in veal herds owned by producers that fatten these calves for this integration on contract. Producers used higher dosages than prescribed by the veterinarian in cohorts with a single caretaker (P < 0.01).CONCLUSIONS:The present study provided detailed information on the intensive antimicrobial use in the white veal industry. Reduction can only be achieved by reducing the number of oral group treatments.

The aim of this study was to determine the link between the characteristics of antimicrobial therapy and occurrence of antimicrobial resistance in Escherichia coli of clinically healthy pigs exposed to antimicrobial treatments.A total of 918 Escherichia coli isolates were obtained from faecal samples, collected from 50 pig herds at the end of the fattening period and susceptibility was tested towards 15 different antimicrobial agents, using the disk diffusion method.The Antimicrobial Resistance Index (ARI) of each isolate was calculated, as the number of antimicrobials to which resistance was found divided by the number of drugs tested.The antimicrobial resistance percentage per class (ARclass) was defined as the percentage of E. coli strains clinically resistant to that specific class.Data on group level antimicrobial use in the sampled herds was collected and quantified as treatment incidences (TI) based on the used daily dose pig (UDDpig) and the animal daily dose pig (ADDpig) (number of pigs treated with one ADDpig or UDDpig/1000 pigs at risk/day).The UDDpig/ADDpig ratio gives an indication of the correctness of dosing.The TIADDpig for group level use was 235.7 per 1000 pigs at risk per day, whereas the TIUDDpig equaled 200.7.This means that in reality, fewer pigs were treated with the same amount of antimicrobials than theoretically expected and thus antimicrobials were generally overdosed.Generalized linear regression analysis showed a significant relation between the TIADDpig and the ARI (p< 0.01), whereas there were no significant links for the TIUDDpig (p> 0.05).Analysis of the antimicrobial resistance for ć-lactam antimicrobials and tetracyclines suggests that the effect of correct or incorrect dosing on resistance development was different for the different antimicrobial classes tested.Besides the amount of administered antimicrobial agents, the frequency of drug administration may play a role in the selection of antimicrobial resistance in commensal E. coli.

Changing an antimicrobial treatment regimen has shown to influence the occurrence of antimicrobial resistance, with regimen consisting of the dose, the treatment interval, the duration of therapy, and the route of administration of the drug. For concentration dependent antimicrobials there is substantial evidence to encourage the use of a high dose, with regular treatment interval and short course to minimise the risk for the selection of resistant mutants. However, in contrast with these first three aspects of the antimicrobial treatment regimen, little attention is currently paid to the influence of route of administration for the probability of selection and spread of resistant strains.

SAMENVATTINGColibacillose is een infectieuze ziekte veroorzaakt door aviaire pathogene Escherichia coli (APEC).Het is nog altijd een van de hoofdoorzaken van grote economische verliezen in de pluimvee-industrie.APEC-stammen zouden ook urineweginfecties bij de mens veroorzaken, maar de literatuur hieromtrent is niet eenduidig.Het stijgende voorkomen van antimicrobiële resistentie zorgt voor groeiende problemen bij de behandeling van de kippen en voor een toenemend risico voor de mens.De nadruk in de bestrijding van colibacillose moet dan ook liggen op de preventie van ziekte-insleep op het pluimveebedrijf met zo weinig mogelijk antibioticumgebruik.

Determining herd- or flock-specific antimicrobial resistance profiles is important to guide therapeutic use of antimicrobials and to assess risk factors for the development and spread of antimicrobial resistance. As such, it is of utmost importance to optimize the sampling strategy for the determination of herd-specific antimicrobial resistance profiles. However, the multitude of prevalences measured at the same time as well as the presence of variation both at the level of the animal and the bacterial population of concern make it impossible to use conventional sample size determination methods. In this article, the use of bootstrapping techniques for sample size determination was explored. In particular, one-stage and two-stage bootstrap samplings were used to determine the optimal number of animals and the optimal number of isolates within one animal. Results show that focus should be on the number of animals sampled rather than on the number of isolates tested within one animal.

Broad-spectrum β-lactamase genes (coding for extended-spectrum β-lactamases and AmpC β-lactamases) have been frequently demonstrated in the microbiota of food-producing animals. This may pose a human health hazard as these genes may be present in zoonotic bacteria, which would cause a direct problem. They can also be present in commensals, which may act as a reservoir of resistance genes for pathogens causing disease both in humans and in animals. Broad-spectrum β-lactamase genes are frequently located on mobile genetic elements, such as plasmids, transposons and integrons, which often also carry additional resistance genes. This could limit treatment options for infections caused by broad-spectrum β-lactam-resistant microorganisms. This review addresses the growing burden of broad-spectrum β-lactam resistance among Enterobacteriaceae isolated from food, companion and wild animals worldwide. To explore the human health hazard, the diversity of broad-spectrum β-lactamases among Enterobacteriaceae derived from animals is compared with respect to their presence in human bacteria. Furthermore, the possibilities of the exchange of genes encoding broad-spectrum β-lactamases - including the exchange of the transposons and plasmids that serve as vehicles for these genes - between different ecosystems (human and animal) are discussed.

SUMMARY The aim of this study was to determine the potential association between housing type and multiple drug resistance (MDR) in Escherichia coli and Enterococcus faecalis isolates recovered from 283 laying-hen flocks. In each flock, a cloacal swab from four hens was collected and produced 1102 E. coli and 792 E. faecalis isolates. Broth microdilution was used to test susceptibility to antimicrobials. Country and housing type interacted differently with the MDR levels of both species. In the E. coli model, housing in a raised-floor system was associated with an increased risk of MDR compared to the conventional battery system [odds ratio (OR) 2·12, 95% confidence interval (CI) 1·13–3·97)]. In the E. faecalis model the MDR levels were lower in free-range systems than in conventional battery cages (OR 0·51, 95% CI 0·27–0·94). In Belgium, ceftiofur-resistant E. coli isolates were more numerous than in the other countries.

AIMS:The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla(TEM-52) -carrying plasmid (lactose-negative mutant of B1-54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. METHODS AND RESULTS:Fresh faeces from a healthy volunteer, negative for cephalosporin-resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed-field gel electrophoresis profiles (PFGE). The avian extended-spectrum β-lactamase-positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1-54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla(TEM-52) -carrying plasmid. CONCLUSIONS:These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. SIGNIFICANCE AND IMPACT OF THE STUDY:The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.

SUMMARY A cross-sectional study on 32 different Belgian broiler farms was performed in 2007 and 2008 to identify risk factors for ceftiofur resistance in Escherichia coli . On each farm, one E. coli colony was isolated from 30 random birds. Following susceptibility testing of 14 antimicrobials, an on-farm questionnaire was used to obtain information on risk factors. Using a multilevel logistic regression model two factors were identified at the animal level: resistance to amoxicillin and to trimethoprim–sulfonamide. On the farm level, besides antimicrobial use, seven management factors were found to be associated with the occurrence of ceftiofur resistance in E. coli from broilers: poor hygienic condition of the medicinal treatment reservoir, no acidification of drinking water, more than three feed changes during the production cycle, hatchery of origin, breed, litter material used, and treatment with amoxicillin. This study confirms that not only on-farm antimicrobial therapy, but also management- and hatchery-related factors influence the occurrence of antimicrobial resistance.

This study explored the prevalence and persistence of acquired antimicrobial resistance in Escherichia coli and Enterococcus faecium from healthy broilers. In 32 broiler farms, cloacal samples were taken during two production rounds, with one production round in between. For 10 of the sampled flocks, samples from the carcasses at the slaughterhouse were also collected. For E. coli, high levels of resistance were found for ampicillin, nalidixic acid, streptomycin, tetracycline, and the combination of trimethoprim and sulfonamide. Over 58% of all the isolates showed resistance to four or more antimicrobial agents. Only 4.8% were fully susceptible for all 14 drugs tested. A remarkably high resistance rate (up to 41%) to ceftiofur was found. The enterococci were frequently resistant to macrolides, tetracycline, and the combination quinopristin/dalfopristin. Over 80% displayed acquired resistance to four or more antimicrobial agents, and 3.9% were fully susceptible for the eight agents tested. Resistance was found to persist over consecutive production rounds. There was a good correlation between results obtained with cloacal samples of the live animals and caecal content samples collected in the slaughterhouse for both E. coli and E. faecium. For E. coli but not for E. faecium, the resistance profile of neck skin isolates was different from that of cloacal isolates.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates were obtained from hospitalized and nonhospitalized patients in Belgium between August 2006 and November 2007. The antimicrobial susceptibility of these isolates was determined and their ESBL genes were characterized. Clonal relationships between the CTX-M-producing E. coli isolates causing urinary tract infections were also studied. A total of 90 hospital- and 45 community-acquired cephalosporin-resistant E. coli isolates were obtained. Tetracycline, enrofloxacine, gentamicin, and trimethoprim-sulfamethaxozole resistance rates were significantly different between the community-onset and hospital-acquired isolates. A high diversity of different ESBLs was observed among the hospital-acquired E. coli isolates, whereas CTX-M-15 was dominating among the community-acquired E. coli isolates (n = 28). Thirteen different pulsed-field gel electrophoresis profiles were observed in the community-acquired CTX-M-15-producing E. coli, indicating that multiple clones have acquired the bla(CTX-M-15) gene. All community-acquired CTX-M-15-producing E. coli isolates of phylogroups B2 and D were assigned to the sequence type ST131. The hospital-acquired CTX-M-15-producing E. coli isolates of phylogroups B2, B1, A, and D corresponded to ST131, ST617, ST48, and ST405, respectively. In conclusion, CTX-M-type ESBLs have emerged as the predominant class of ESBLs produced by E. coli isolates in the hospital and community in Belgium. Of particular concern is the predominant presence of the CTX-M-15 enzyme in ST131 community-acquired E. coli.